The main goal of this project is to investigate the evolutionary constraints that govern the natural selection of partially collapsed, flexible proteins and protein domains. To accomplish this goal we have selected a model system based on a conserved flexible linker from the 70kDa subunit of replication protein A (RPA70). We will use this model system to determine if and how natural selection works to preserve the structure and function of partially collapsed, flexible proteins and protein domains through the following specific aims: 1) Characterize the structure, dynamics, and function of the linkers from several RPA70 homologues and determine the functional consequences of linker swapping; 2) Investigate the allowable sequence space of the RPA70 flexible linkers by experimental evolution and by conducting a broad survey of eukaryotic linker sequences; 3) Use the results of specific aims 1 and 2 to formulate rules that permit the identification and alignment of flexible sequences as well as the de novo design of sequences that function as linkers. While the presence and importance of partially collapsed, flexible proteins and protein domains in nature as well established, this proposal marks the first systematic empirical investigation into their evolutionary constraints and patterns.